CN113846027B - Metal-resistant copper bacteria strain and application thereof - Google Patents
Metal-resistant copper bacteria strain and application thereof Download PDFInfo
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- CN113846027B CN113846027B CN202110729266.XA CN202110729266A CN113846027B CN 113846027 B CN113846027 B CN 113846027B CN 202110729266 A CN202110729266 A CN 202110729266A CN 113846027 B CN113846027 B CN 113846027B
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- 229910052802 copper Inorganic materials 0.000 title claims abstract description 44
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- 230000000694 effects Effects 0.000 claims description 31
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- 239000011574 phosphorus Substances 0.000 claims description 22
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- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses a metal-resistant copper fungus strain named as copper fungus (cupravidus) metallidurans) S-8-2 strain with preservation number of CGMCC NO. 20011. The strain can be used for preparing plant growth promoters and soil restoration agents under various heavy metal stresses.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a metal-resistant copper bacteria strain and application thereof.
Background
Most of the soil in China is polluted by multiple metals to different degrees, and for a large number of agricultural lands polluted by heavy metals, development of soil restoration technology meeting the requirements of agricultural production is urgently needed. Soil microorganisms play an important role, wherein plant rhizosphere growth promoting bacteria (Plant growth promoting rhizobacteria, PGPR) exist on plant rhizosphere and root surfaces, physical and chemical properties of soil can be improved through phosphate dissolving, nitrogen fixation and the like, toxic substances in the soil are reduced to be harmless, nutrient circulation is promoted, the soil is symbiotic with plants, nutrition is provided for plant growth, plant growth can be promoted through secretion of plant hormones such as indoleacetic acid (IAA), cytokinin, gibberellin and the like, plant pathogenic bacteria are inhibited, the contaminated soil is repaired while plant resistance is improved, and functions such as soil quality health are maintained. The rhizosphere soil of the dominant plant golden buckwheat of the heavy metal antimony resistance around the abandoned mine is screened to obtain a strain of rhizosphere-promoting bacteria copper greedy fungus, the strain has a phosphorus dissolving effect, the phosphorus which is difficult to absorb and utilize by plants is converted into a form which can be absorbed and utilized for plant growth and development, meanwhile, the strain can generate growth hormone IAA to directly promote plant growth, and the screened microorganism with the capability of producing IAA can provide an initial strain for development of growth-promoting biofertilizer and the like.
Therefore, PGPR which is resistant to heavy metals and can promote germination of crop seeds is separated and screened, biological characteristics of the PGPR and the growth promotion effect of the PGPR on crops in heavy metal pollution areas are researched, and the PGPR is definitely helpful for agricultural production in heavy metal pollution areas, ensures green sustainable development of agriculture and improves soil ecological environment.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provides a metal-resistant copper bacteria strain and application thereof.
In order to achieve the above purpose, the technical scheme provided by the application is as follows:
the strain of the metal-resistant copper bacteria is named as a metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 strain, and is preserved in China center for type culture Collection (address: university of Wuhan, china) with a preservation number of CGMCC NO.2091 at 10-month 19 of 2020. The 16S rDNA sequence of the strain is shown as SEQ ID NO. 1.
The metal-resistant copper fungus strain can be used for preparing plant growth promoters under various heavy metal stresses.
Preferably, the plurality of heavy metals includes at least one of cadmium, arsenic, lead, antimony (As, sb, pb, cd), i.e., the strain of the genus greedy metal is resistant to a plurality of heavy metals, such as cadmium, arsenic, lead, antimony, and the like.
Preferably, the growth promoter is a preparation with a phosphorus dissolving effect, namely, the metal-resistant copper bacteria strain has a phosphorus dissolving effect under heavy metal stress, and can convert phosphorus which is difficult to absorb and utilize by plants into phosphorus which can be used for soil phosphate fertilizers, such as quick-acting phosphorus.
Preferably, the growth-promoting agent is a formulation capable of synthesizing auxin IAA using tryptophan.
Preferably, the growth promoter is an agent capable of secreting ACC deaminase, namely, the metal-resistant copper bacteria strain can regulate the level of plant endogenous ethylene by degrading ethylene precursor substance 1-aminocyclopropane-1-carboxylic Acid (ACC), reduce the influence on crops under heavy metal stress and promote the growth and development of the crops.
Preferably, the growth promoter is a broad-spectrum antibiotic formulation.
Preferably, the growth promoter is a crop rape seed germination promoter.
Preferably, the growth promoter is an antioxidant system activity promoter, namely, the metal-resistant copper fungus strain has an effect of promoting the antioxidant system activity of seeds in the rape seed germination process under heavy metal stress.
The metal-resistant copper fungus strain can also be used for preparing soil restoration agents, namely, the metal-resistant copper fungus strain can be combined with crop planting for restoring soil in heavy metal farmland polluted areas.
The application is further described below:
the metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 provided by the application are obtained by screening and separating rhizosphere soil of wild buckwheat which is a dominant plant of a waste mine of a Hunan tin mine for the first time, and the strain has high-concentration heavy metal antimony arsenic resistance, and the detection result shows that the strain is a novel metal-resistant copper bacteria.
The metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 provided by the application can dissolve and utilize calcium phosphate in a culture medium to secrete small molecular organic acid, has the characteristics of producing growth hormone IAA, producing ACC deaminase, resisting broad-spectrum antibiotics and the like, can resist high-concentration heavy metals, on one hand, convert the heavy metals in soil, relieve the heavy metal stress of crops, change the valence state and morphology of polyvalent As and Sb, and reduce the accumulation of the polyvalent As and Sb in the roots of the crops, thereby improving the nutrient content and absorption condition of the soil, promoting the plants to absorb more nutrient substances and promoting the growth of the plants. On the other hand, the plant growth promoting agent has the characteristic of promoting growth and can promote the germination and growth of crop seeds.
The metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 provided by the application can synthesize IAA by taking tryptophan as a precursor, can be directly utilized by crops due to the fact that the tryptophan is adsorbed on the surfaces of crop seeds and root systems, and can also stimulate the growth and proliferation of cells in the crops under the combined action of IAA which is generated in the crops, so that the growth and development of the root systems of the crops in heavy metal polluted farmland soil are promoted, and the moisture and nutrients in the soil are effectively absorbed. And may also be involved in the regulation of other vital activities within the crop.
The metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 provided by the application has an obvious promotion effect on rape seed germination under high-concentration heavy metal antimony stress, and has an obvious enhancement effect on the activity of an antioxidant system of rape seeds.
Drawings
FIG. 1 shows colony morphology of the copper-tolerant bacteria (Cupriavidus metallidurans) S-8-2 of the application in CDM medium;
FIG. 2 is a high performance liquid analysis chart of organic acid production from fermentation broth of copper (Cupriavidus metallidurans) S-8-2 strain containing 10mM Sb 3+ The liquid NBRIP liquid culture medium is subjected to liquid phase analysis after continuous fermentation for 2 days under the conditions of 28 ℃ and 120rmp/min, and the sample injection amount is 0.02ml;
FIG. 3 is a phosphorus dissolution standard curve for determining phosphorus dissolution capacity;
FIG. 4 is an IAA standard curve for determining the IA A production capacity using the Sackowski chromogenic method;
FIG. 5 is a standard curve for the determination of alpha-ketobutyric acid concentration by dinitrophenylhydrazine method;
FIG. 6 is a graph showing the effect of a strain of copper (Cupriavidus metallidurans) S-8-2 resistant to stress with heavy metal antimony on the root development of canola seeds.
Detailed Description
The application will now be described in further detail with reference to specific examples, which are intended to illustrate, but not to limit, the application.
Materials and reagents used in the examples described below, unless otherwise indicated, were all commercially available. The experimental methods used in the following examples are conventional methods unless otherwise specified.
Example 1 isolation and identification of strains
1.1 isolation of copper (Cupriavidus metallidurans) S-8-2 resistant bacteria
The separation of the metal-tolerant copper bacteria (Cupriavidus metallidurans) S-8-2 comprises the steps of sampling, enriching and preliminary screening, and re-screening, wherein the method comprises the following steps:
1.1.1 sampling
The sample is taken from rhizosphere soil of a waste mine of Hunan tin mine, root systems of plants are dug out along the growth direction of the root systems at intervals of 120 degrees around wild buckwheat plants, a large amount of non-rhizosphere soil carried by the root systems is removed by gently shaking off the root systems, then the soil attached to the surfaces of the plant root systems is brushed in a sterile bag by a sterile brush, marks are made, and the plants are transported back to a laboratory at normal temperature and kept in a refrigerator at 4 ℃ for standby.
1.1.2 enrichment and preliminary screening
Weighing 100g of soil sample into sterilized glass bottle, adding 1ml of potassium antimoniate C with concentration of 100mM 8 H 4 K 2 O 12 Sb 2 .3H 2 O, gently stirring, placing in a 28 ℃ incubator, enriching and culturing for 7d, and adding sterile water during enrichment process to ensure that the sample keeps original humidity.
Weighing the enriched soil sample 10g, culturing in triangular flask containing 90ml sterile physiological saline at 28deg.C shaking table at 160r/min for 20min, and gradient diluting to 10 -3 ，10 -4 ，10 -5 0.1ml of each gradient of the diluted solution was applied with 100 mu M C 8 H 4 K 2 O 12 Sb 2 .3H 2 On CDM selective plates of O, 3 plates were coated for each dilution, and after culturing in a constant temperature incubator at 28℃for 7d, the presence or absence of single colonies was observed, and single colonies were picked and streaked multiple times to ensure pure culture.
1.1.3 double sieves
The strain obtained in 1.1.2 was cultured in LB medium at 30℃and 120rpm for 24 hours. Then IAA examination is carried out on the fermentation liquor, the fermentation liquor is taken and diluted according to the gradient and is coated on the fermentation liquor containing 5mM Sb 3+ CDM solids of (2)In the culture medium, the strain with higher heavy metal resistance is obtained by reversely culturing for 3 days in a 30 ℃ incubator and selecting larger strain for multiple separation passages. 1.2 identification of copper (Cupriavidus metallidurans) S-8-2 resistant bacteria
And (3) carrying out a series of physiological and biochemical identification on the pure culture strain obtained by the separation and purification, and carrying out DNA extraction, amplification and sequencing of 16S rDNA.
1.2.1 As shown in FIG. 1, the strain forms yellowish, convex, smooth-surfaced, clean-edged oval or circular colonies on CDM solid medium.
Microscopic examination showed short rod-shaped, flagellum-free, spore-free gram-negative bacteria, and other test items are shown in table 1.
TABLE 1 physiological and biochemical characteristics of S-8-2 Strain
Note that: in the above table, + indicates a positive reaction, present or present, -indicates a negative reaction, absent or absent
1.2.2 amplification of 16S rDNA with primers 27F and 1492R, the sequences of the primers are as follows:
27F：5′-AGAGTTTGATCATGGCTCAG-3′(SEQ ID NO.2)；
1492R：5′-TAGGGTTACCTTGTTACGACTT-3′(SEQ ID NO.3)。
the PCR amplification conditions were 95℃for 5min,95℃for 30s, 58℃for 30s, 72℃for 90s, 72℃for 7min, and 35 cycles.
The PCR amplified product was sequenced, and the sequencing result was shown as SEQ ID NO.1, and the amplified length of the 16SrDNA sequence of the S-8-2 strain was 2481kb.
Through homology comparison, the strain has 98.71 percent of similarity with Cupriavidus metallidurans CH (NCBI access No. NR_ 074704.1), combines the morphological and physiological biochemical characteristics of the S-8-2 strain, identifies the strain as a strain of Cupriavidus metallidurans genus, is named as copper-clad fungus (Cupriavidus metallidurans) S-8-2, and is preserved in China Center for Type Culture Collection (CCTCC) (address: university of Wuhan, china) with a preservation number of CCTCC NO: 2091.
Example 2: determination of organic acid production type and determination of metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 under heavy metal antimony stress
S-8-2 Strain in 10mM Sb 3+ The fermentation was continued for 3 days at 28℃and 120rmp/min, and then the organic acid in the fermentation broth was analyzed by High Performance Liquid Chromatography (HPLC). The analysis conditions were a chromatographic column C18 (250 mm. Times.4.6 mm,5 μm); ultraviolet detection wavelength 215nm; the flow rate is 0.4ml/min, and the sample injection amount is 20 μl; the mobile phase was dipotassium hydrogen phosphate (analytically pure, SIGMA) at pH 2.5. The analysis result shows that the S-8-2 can effectively utilize glucose in the culture solution to be converted into propionic acid, so as to dissolve calcium phosphate in the culture solution, dissolve the metal calcium chelate and release quick-acting phosphorus.
As shown in FIG. 2, the metal-resistant copper bacteria (Cupriavidus metallidurans) of the application has the highest propionic acid production, citric acid and glucuronic acid production.
Example 3: determination of phosphorus dissolving capability of metal-tolerant copper bacteria (Cupriavidus metallidurans) S-8-2 under heavy metal antimony stress
In the presence of 10mM Sb 3+ The liquid NBRIP liquid culture medium of (1) is provided with no bacteria, no bacteria and no Ca 3 (PO 4 ) 2 Control, 3 replicates per treatment. The treatment group is inoculated with 1% of S-8-2 bacterial liquid in logarithmic phase, and after inoculation, the culture is carried out for 72 hours under the conditions of 28 ℃ and 120rmp/min, the culture liquid is centrifuged at 8000rpm for 5min, the supernatant is taken, and after passing through a 0.22pm pore-size filter membrane, the quick-acting phosphorus concentration is measured by a molybdenum blue method. The results show that the effective phosphorus concentration of the inoculated S-8-2 strain is 501.63 +/-22.47 mg/l, the sterile control group is only 3.05+/-0.11 mg/l, and the activation capacity of the treatment group to Ca-P is 164 times that of the sterile control group.
As shown in FIG. 3, the phosphorus dissolving capacity of the copper (cupravidus sp) S-8-2 under heavy metal antimony stress is shown as the effective phosphorus concentration of 501.63 strych 22.47mg/l.
Example 4: determination of the content of the synthetic growth hormone IAA of the copper-tolerant bacterium (Cupriavidus metallidurans) S-8-2
By using Salkowski methodQuantitative IAA production detection of S-8-2 strain, culturing in LB liquid medium containing 0.5g/l tryptophan at 28deg.C under 150rmp for 48 hr, and measuring OD with spectrophotometer 535 IAA standard curves were used to calculate IAA for each treatment. Experiments find that 2mM Sb 3+ And 10mM Sb 3 Stress has no great influence on the IAA production capacity of the strain, the IAA concentration is not greatly different from that of a control group, is basically stabilized at about 94.75 mug/ml, is cultured for about 36 hours, the IAA production concentration is saturated, and the IAA production concentration is not increased after continuous culture.
As shown in FIG. 4, the amount of IAA synthesized by the copper (cupravidus sp) S-8-2 resistant bacterium of the present application was 94.75. Mu.g/ml.
Example 5: ACC deaminase Activity detection of copper-resistant bacteria (Cupriavidus metallidurans) S-8-2
After culturing the strain S-8-2 in TSB culture medium at 28℃and 160rmp in the dark for 12 hours, the bacterial pellet was collected by centrifugation. The bacterial strain was subjected to washing centrifugation 2 times with Tris-HCl buffer (0.1 mol/l, pH 7.6), then the cell pellet collected by centrifugation was resuspended with 600. Mu.l of Tris-HCl buffer (0.1 mol/l, pH 8.5), and 30. Mu.l of toluene was added thereto and then rapidly shaken for 30 seconds to disrupt the cells to obtain ACC deaminase, the ACC deaminase activity of the strain was measured using the 2, 4-dinitrophenylhydrazine method, the absorbance value thereof was measured at 540nm, and the ACC deaminase activity of the strain was calculated using the standard curve of alpha-butanoic acid.
As shown in FIG. 5, the S-8-2 strain had ACC deaminase activity, and its enzymatic activity reached 389.06. Mu. Mol/(mg.h).
Example 6: minimum tolerance concentration (MIC) of copper-tolerant bacteria (Cupriavidus metallidurans) S-8-2 to different heavy metals
The Minimum Inhibitory Concentration (MIC) refers to the minimum concentration at which a substance having an inhibitory effect inhibits the production of a strain of interest. The experiment was carried out by determining the highest concentration of the strain S-8-2 at 7 heavy metals.
The activated metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 are streaked and inoculated on LB culture medium plates containing heavy metals with different concentrations, and after culturing for 3 days in a biochemical incubator at 28 ℃, whether the bacteria grow or not is observed. As a result, strain S-8-2 was able to tolerate various strains as shown in Table 2Heavy metals. Minimum inhibitory concentrations of Sb +3 (10mM)、As +3 (40mM)、Pb +2 (4mM)、Cd +2 (5mM)、Cr +6 (6mM)、Cu +2 (8mM)、Zn +2 (25mM)。
TABLE 2 growth of S-8-2 Strain on LB Medium containing different concentrations of heavy metals
Note that one representation in the table above does not grow; + stands for growth
Example 7: sensitivity of copper-resistant bacteria (Cupriavidus metallidurans) S-8-2 to broad-spectrum antibiotics
The bacterial liquid cultured to the logarithmic phase of 200uL is coated on a TSA solid culture medium, 10 drug sensitive paper sheets with antibiotics with different concentrations are placed on a flat plate, the growth condition of the drug sensitive paper sheets is observed and transferred for 2 times, and the experimental results are verified, and the results are shown in Table 3. S-8-2 has broad-spectrum antibiotic sensitivity, and is beneficial to further realizing the application of S-8-2 in farmland soil remediation.
TABLE 3 determination of sensitivity of S-8-2 Strain to antibiotics
Note in the table above +: representing sensitivity and degree-: representing insensitivity
Example 8: growth promotion by stress of copper (Cupriavidus metallidurans) S-8-2 antimony
Growth promotion by 10mM Sb 3+ The experiment of the metal-tolerant copper bacteria (Cupriavidus metallidurans) S-8-2 on plant seed germination and growth promotion under the ion stress is embodied. The specific process is as follows:
(1) filling the filter paper in a culture dish, spreading the filter paper on the bottom of the dish as much as possible, sterilizing for 20min at 121 ℃ by wet heat, and drying at 70 ℃ for later use.
(2) Preparation of bacterial suspension: inoculating the strain into LB culture medium, culturing at 37deg.C and 200rpm for 18 hr, centrifuging to collect thallus, and suspending with distilled waterFloating, adding distilled water to OD 600 =0.8, the bacterial suspension is about 10 8 CFU/ml。
(3) Seed surface disinfection and seed soaking: selecting plump rape seeds, cleaning with sterile distilled water for three times, cleaning with 75% alcohol for two times, soaking with 75% alcohol for 2min, and cleaning with sterile distilled water for three times for use. Soaking seeds which are sterilized by using the prepared bacterial suspension, wherein distilled water is used for a control group; after seed soaking for 4 hours, the next experiment was performed.
(4) 3ml of 1/4Hoagland broth (fully soaked filter paper) was added to the petri dish; adding C under the above conditions 8 H 4 K 2 O 12 Sb 2 Solution of Sb 3+ The concentration was kept at 10mM; equivalent distilled water was added to the control group. Each culture dish is filled with 20 immersed seeds, and the seeds are placed in a climatic chamber under the culture condition that the temperature is 25 ℃, and the light/dark is 14h/10h, and the culture is carried out for 7d. And a proper amount of distilled water is required to be supplemented every day to make up the evaporated water, the germination condition is observed at the same time every day, and the germination index is recorded. And (3) calculating seed germination rate after 7 days, measuring biomass indexes such as root length, stem length, fresh weight, dry weight and the like, and preliminarily judging the growth promotion effect of the strain S-8-2 on the rape under the stress of antimony.
As shown in FIG. 6, the test strain of the application, copper (Cupriavidus metallidurans) S-8-2, has a growth promoting effect on rape seeds under antimony stress.
As shown in table 4, 3 germinated seeds were randomly selected as one biological repetition in the control group and the experimental group, respectively, and biomass measurement was performed in combination with a root system scanner, and chlorophyll content, root system activity, soluble protein and soluble sugar content were measured by acetone extraction method, TTC method, coomassie brilliant blue G-250 method and anthrone method, respectively. The result shows that the biomass of the treatment group is obviously increased (P is less than 0.01) compared with that of the control group, the germination rate is increased by 31.9%, the fresh weight stem and root are respectively increased by 50.00% to 146.67%, and the dry weight stem and root are respectively increased by 44.44% to 150.00%; the root length of the root system is increased by 254.76%, the root surface area is increased by 220.81%, and the total root tip number of the root system is increased by 200.00%. In addition, the root system activity, total chlorophyll, root system soluble protein, stem soluble protein and soluble sugar of the seed after germination of the inoculated strain are also obviously increased compared with the control group, and the increase amounts respectively reach 70.3%, 53.3%, 208.33%, 133.33%, 45.7% and 61.2%. And the difference of soluble sugar of root systems between the two groups of treatments is not obvious. The inoculation of the metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 can promote the germination and growth of seeds under the stress of high-concentration heavy metal antimony, and improve part of the physiological activity of the seeds.
TABLE 4S-8-2 influence on physiological index of rape seed germination under antimony stress
* The treatment group was significantly different from the control group (P < 0.05)
5 germinated seeds are randomly adopted as biological repetition in a control group and an experimental group respectively, and Peroxidase (POD), superoxide dismutase (SOD) and Catalase (CAT) activities of the germinated seeds are measured by adopting a guaiacol method, an NBT reduction method and a potassium permanganate titration capacity method, wherein 3 repetitions are set for each index, and the germination is carried out by dividing stems and root systems into 2 parts. The results of antioxidant enzyme activity of germinated seeds show (Table 5), after inoculation, the SOD enzyme activity of the root system part of the treated seeds is obviously increased by 93.0% and 30.4% compared with that of the control group, and only CAT enzyme activity of the overground part is obviously increased by 47.0%. The strain S-8-2 can obviously improve 1-2 of 3 antioxidant enzyme activities of seed germination under high-concentration heavy metal antimony stress, and particularly has larger influence on the antioxidant enzyme activity of the underground root system part of the plant.
TABLE 5S-8-2 Effect on antioxidant enzyme Activity of rape seed germination under antimony stress
* The treatment group was significantly different from the control group (P < 0.05)
In conclusion, the metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 can resist heavy metals (Sb, as, cr, cd, pb, zn, cu), so that the toxicity of heavy metals in soil can be reduced by absorbing the heavy metals and converting the valence states of the heavy metals, and the growth of plants can be promoted. The organic acid small molecule can be generated by dissolving and utilizing calcium phosphate under the stress of heavy metal, insoluble phosphorus is efficiently dissolved, soluble phosphorus is released, the quick-acting phosphorus content in soil is increased, the stress of plant phosphorus deficiency can be relieved, and the plant growth is promoted. Meanwhile, the composite material has broad-spectrum resistance to antibiotics in soil, has ACC deaminase activity, can reduce the toxicity of heavy metals to plant cells, can synthesize indoleacetic acid, promote plant growth, and can help to improve farmland soil by converting and resolving the heavy metals in soil and promoting the increase of quick-acting phosphorus content. Therefore, the above-described action has a commonality in promoting plant growth, and therefore can be applied to plant growth promotion as a wide range of growth-promoting bacteria.
The metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 can promote seed germination under heavy metal stress from multiple aspects, and the metal-resistant copper bacteria (Cupriavidus metallidurans) S-8-2 can treat rape seed germination rate under heavy metal antimony stress, has obvious promotion effect on biomass of stems and roots, can obviously improve oxidation resistance, can also obviously improve the quick-acting phosphorus content of soil, and can be applied to preparation of rape growth promoting preparations or microbial fertilizers.
The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the application and its practical application to thereby enable one skilled in the art to make and utilize the application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.
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Claims (8)
1. A strain of a metal-resistant copper bacterium, characterized in that the metal-resistant copper bacterium strain is named metal-resistant copper bacterium (cupravidus) metallidurans) S-8-2 strain with preservation number of CGMCC NO. 20011.
2. The use of a strain of a metal tolerant cuprum as claimed in claim 1 for the preparation of plant growth promoters under stress of a plurality of heavy metals including at least one of cadmium, arsenic, lead, antimony.
3. The use according to claim 2, wherein the growth promoter is a formulation having a phosphorus dissolving effect.
4. The use according to claim 2, wherein the growth-promoting agent is a preparation capable of synthesizing auxin IAA using tryptophan.
5. The use of claim 2, wherein the growth-promoting agent is an agent capable of secreting ACC deaminase.
6. The use according to claim 2, wherein the growth promoter is a crop-promoting canola seed germination agent.
7. The use according to claim 2, wherein the growth promoter is an antioxidant system activity promoter.
8. Use of a strain of a metal-tolerant cupreous strain according to claim 1 for the preparation of a soil remediation agent.
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Application publication date: 20211228 Assignee: Hunan Shuotai Agricultural Comprehensive Development Co.,Ltd. Assignor: Hunan University of Humanities, Science and Technology Contract record no.: X2023980052631 Denomination of invention: A strain of metal resistant copper loving bacteria and its application Granted publication date: 20230822 License type: Common License Record date: 20231218 |